Emerging evidence indicates that renal cystic disease in both humans and mouse models involves a multigenic pathway in which the disease-susceptibility genes act by cellular recessive mechanisms. These genes also appear to play critical roles in renal differentiation and maturation. The cpk mouse was the first polycystic kidney disease (PKD) model to be described and as such, it has been the most extensively characterized. While these studies have significantly contributed to our understanding of renal cystic disease, they have been conducted primarily in mice with advanced renal cystic disease. Therefore, the critical relationship between the normal epithelial differentiation and the initial events of renal cyst formation remains to be elucidated and the molecular basis for the cpk phenotype is as yet unknown. The ultimate goals of the PI are (1) to characterize the role of the cpk gene product in renal development by determining the molecular pathways in which it functions, and (2) to elucidate how the loss of function of this gene causes renal cyst formation. In this grant application, the PI proposes to establish the biologic and molecular framework for these investigations. The proposal is divided into two complementary projects. First, in Aim 1, they propose to test whether the prevailing hypotheses regarding renal cystogenesis apply to the genetically-defined, fetal cpk model that they have recently characterized. Second, they propose to clone the cpk gene. Aim 2 deals with the construction of a complete transcript map of the critical cpk interval. Aim 3 deals with the identification of the cpk gene from the transcript map generated. In Aim 4 they propose to examine the temporal and spatial expression of the cpk gene during the stages of renal organogenesis, e.g. induction, acquisition of stem cell character, fate determination, condensation, epitheliogenesis, polarization, and maturation. Significance: The cpk mutation represents one of a number of recessive +____________ mutations causing PKD in the mouse. Like most other spontaneous mutations, the severe kidney disease of this mutant demonstrates the importance of the normal cpk counterpart in renal function. Given that the mechanism of cystogenesis is not well understood, despite the information provided by the cloning of PKD1 and PKD2. There is an increased need for discovering new genes in mouse whose deregulation leads to renal cystogenesis. The preliminary work from the laboratories of the principal investigator and the collaborators has resulted in the placement of the cpk mutation to a region between D12Mit218 and D12Mit105 of mouse Chr 12. Based on recombination frequency the two flanking markers were placed 0.26 cM apart (approximately 5 X 10 exp 5 bp). The physical map of the cpk critical region is being generated. Based on the fine mapping data the human cpk homologue maps to Chr 2p24-2p25. The physical map is being generated. Approach: Specific Aim 1: Examine the prevailing hypotheses regarding renal +________ cystogenesis in fetal cpk mouse model. a) disruption in basement membrane 1 PATHOLOGY A SS 3 R01 DK55534-01A1 June, 1999 Guay-Woodford, Lisa M formation. b) dysregulation of cell proliferation and apoptosis. c) aberrant expression of the epidermal growth factor receptor (EGFR), polycystin, and polycystin-2. The hypothesis is that the loss of function of the cpk gene interrupts the terminal phases of renal tubulo-epithelial differentiation. They speculate that the proximate event in renal cystogenesis may be a dysregulation of basement membrane composition with subsequent dysregulation of cell-matrix interactions, cell proliferation, and apoptosis that occur in a hierarchical order. In order to test this they plan to examine the expression of entactin-1, laminin gamma&#61472;1, polycystin-1, polycystin-2 and EGF-R in E15 control (+/+) and cpk/cpk kidneys. They also propose to analyze the cell proliferation and apoptosis in the same. The E15 kidney was chosen since light microscopy revealed tubular dilation only in pups homozygous for B6 alleles. This experiment will provide answers with respect to the altered events in the E15 diseased kidney due to the loss of the cpk gene. However, in order to either confirm or refute the hierarchical order speculated it may be important to study the developmental profile of all the factors proposed during cystogenic process (Time points earlier and later than E15 have to be considered). Specific Aim 2: Isolate all expressed sequences from the cpk candidate interval and construct a complete transcript map of the interval. Based on the Whitehead/MIT database a YAC contig that spanned the 0.26cM interval was generated. Presently they have almost generated a BAC contig of this region. Unfortunately the interval is rather large (750kb). The chance of cutting down this interval considerably is rather remote. Since, only two recombination events in this interval were noted previously and the supplemental data suggests that the SP6 end of BAC-358F10 does not cross the distal recombination event in BC65. In light of their latest results, the identification of additional polymorphic markers to further refine the recombination breakpoints of the cpk interval can take a backseat. The identification of all the expressed sequences from the cpk interval must take priority. As the PI has pointed out Exon trapping, Hybrid cDNA selection and EST database analysis are the ways to go. The use of shot-gun sequencing is not an economical approach especially with a region as big as 750kb (requiring 8,000-12,000 sequencing). Specific Aim 3: Analyze candidate transcripts and identify the cpk gene. The methods proposed by the investigator to analyze the coding part of the candidate genes isolated are logical and well thought out. However, no methods have been discussed to detect mutations in the promoter or intronic region. The in vitro functional complementation assay proposed to test the candidate cpk +__ _____ gene is not established at this juncture. Specific Aim 4: Characterize the temporal and spatial expression of the cpk gene in the mouse fetal kidney. The experiments designed to analyze the expression of the cpk gene in mouse fetal kidney are described well. Innovation: The project does employ a number of new methods and approaches. +___________ 1 PATHOLOGY A SS 4 R01 DK55534-01A1 June, 1999 Guay-Woodford, Lisa M However the project does not challenge any existing paradigms or develop new methodologies. This does not however weaken the application. Investigator: Dr. Guay-Woodford appears to have the technical and intellectual +____________ skills to accomplish the cloning of the cpk gene. Environment: The environment at University of Alabama School of Medicine +___________ appears to be conducive for the research proposed by the investigator. OVERALL EVALUATION: This proposal represents important steps that have to be +__________________ taken to further our understanding of the cpk locus. However the principal weakness of this proposal is Aim 1, the experiments designed do not answer the questions proposed by the PI. As already mentioned a more detailed experimental protocol is required to confirm or refute the speculations. Thus, it would be better to concentrate on the cloning of cpk gene.